Functional and molecular aspects of ion channels in macrophages

Monocytes/macrophages play very important roles in innate and adaptive immunity. Ion channels are small molecules embedded in the cell membrane and they play fundamental roles in cell function. Both topics have been extensively studied in isolation, however the role of ion channels in macrophage function is far less understood. In this thesis, the functional and molecular aspects of two ion channels expressed in macrophages, Kor, a potassium channel, and CLIC1, a chloride channel were studied. The biological function of Kor and CLIC1 in activated human macrophages was examined using ion channel blockers. In addition, the role of CLIC1 in the cell cycling of CHO-K1 cells was also investigated. The in vitro studies showed that Kor and CLIC1 are involved in cytokine production by PMA-activated human macrophages and that CLIC1 is also involved in the cell cycling of CHO-K1 cells. Despite providing interesting data, the results of the in vitro studies were difficult to interpret due to the uncertain specificities of the Cl- channel blockers. Therefore, to understand the biological role of CLIC1 in vivo, a gene targeting experiment was performed to create a CLIC1 knock-out (KO) mouse. This involved cloning the mouse CLIC1 gene, making a targeting vector, producing targeted ES cells, and generating a CLIC1 knock-in (KI) mouse which carries a flag tag at the N-terminal and three loxP sites in the targeted locus. Crossing the CLIC1 KI mouse with the TNAP-Cre mouse, a strain over expressing Cre recombinase under a TNAP promoter, a CLIC1 KO mouse was generated. The initial phenotype analysis showed no major development or growth abnormality in the CLIC1 KO mouse. Instead, hyperplasia of megakaryocytes and possible erythroid cells in the spleen and bone marrow was observed suggesting some degree of abnormality in the haematopoeitic system. Furthermore, a comparison of wild type mice with the CLIC1 KO mouse showed that CLIC1 protein expresses at high levels in monocytes, lymphocytes, platelets, and tissue macrophages of normal animals tissues, such as spleen, kidney (mesangial cell), and liver (kupffer cells). This further indicates that CLIC1 may play a significant role in regulating functions of platelets, lymphocytes, and specially tissue macrophages. More extensive studies can now be performed on the CLIC1 KO mouse to clarify the biological function of CLIC1. In summary, the generation of the CLIC1 KO mouse provides a valuable model to study the biological function of CLIC1 both in vivo and in vitro

Apoptosis-inducing effect of 6,7-dimethoxy-4'-hydroxy-8- formylflavon from Nicotiana tabacum L leaf in human hepatoma HepG2 cells via activation of mitochondriamediated apoptotic pathway

Purpose: To study the anti-proliferative and apoptotic influences of 6,7-dimethoxy-4'-hydroxy-8- formylflavon (DHF) from the leaves of Nicotiana tabacum L. in human hepatoma HepG2 cells, and the underlying mechanisms.Methods: The anti-proliferative effect of DHF (10 - 50 μg/mL) on HepG2 cells was assessed by CCK-8 assay. The pro-apoptotic effect of DHF (10, 20 and 30 μg/mL) on HepG2 cells was investigated via flow cytometry, while the mechanisms involved were studied using western blot. Xenograft assay was employed for determination of the in vivo effect of DHF (40 mg/kg/day) on HepG2 cell-induced tumor.Results: The proliferation of HepG2 cells was inhibited by DHF (IC50 = 25.87 μg/mL) due to apoptosis. In addition, xenograft assay revealed that HepG2 cell-induced tumor growth was significantly suppressed by DHF (p < 0.05 or 0.01) without any effects on mice body weights. The expressions of Survivin and Bcl-2 proteins were significantly decreased, while those of Bax, c-caspase-9, and ccaspase- 3 proteins were significantly increased by DHF (p < 0.05 or 0.01), leading to increase in cytoplasmic levels of Smac and cytochrome c proteins.Conclusion: The underlying mechanism DHF-mediated apoptotic changes in HepG2 cells in vitro and in vivo involves induction of the mitochondrial pathway of apoptosis. Thus, DHF is a good drug candidate for the development of an effective therapy for liver cancer.Keywords: 6,7-Dimethoxy-4'-hydroxy-8-formylflavon, HepG2 cells, Hepatoma, Mitochondria, Apoptosis, Bax, Cytochrome C, Survivi

Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies

A signal-seeking Phase 2 study of olaparib and durvalumab in advanced solid cancers with homologous recombination repair gene alterations

Purpose: To determine the safety and efficacy of PARP plus PD-L1 inhibition (olaparib + durvalumab, O + D) in patients with advanced solid, predominantly rare cancers harbouring homologous recombination repair (HRR) defects. Patients and methods: In total, 48 patients were treated with O + D, 16 with BRCA1/2 alterations (group 1) and 32 with other select HRR alterations (group 2). Overall, 32 (66%) patients had rare or less common cancers. The primary objective of this single-arm Phase II trial was a progression-free survival rate at 6 months (PFS6). Post hoc exploratory analyses were conducted on archival tumour tissue and serial bloods. Results: The PFS6 rate was 35% and 38% with durable objective tumour responses (OTR) in 3(19%) and 3(9%) in groups 1 and 2, respectively. Rare cancers achieving an OTR included cholangiocarcinoma, perivascular epithelioid cell (PEComa), neuroendocrine, gallbladder and endometrial cancer. O + D was safe, with five serious adverse events related to the study drug(s) in 3 (6%) patients. A higher proportion of CD38 high B cells in the blood and higher CD40 expression in tumour was prognostic of survival. Conclusions: O + D demonstrated no new toxicity concerns and yielded a clinically meaningful PFS6 rate and durable OTRs across several cancers with HRR defects, including rare cancers

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution.

Abstract CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathioneS-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1–90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes

Proton-Boron Fusion Yield Increased by Orders of Magnitude with Foam Targets

A novel intense beam-driven scheme for high yield of the tri-alpha reaction 11B(p,{\alpha})2{\alpha} was investigated. We used a foam target made of cellulose triacetate (TAC, C_9H_{16}O_8) doped with boron. It was then heated volumetrically by soft X-ray radiation from a laser heated hohlraum and turned into a homogenous, and long living plasma. We employed a picosecond laser pulse to generate a high-intensity energetic proton beam via the well-known Target Normal Sheath Acceleration (TNSA) mechanism. We observed up to 10^{10}/sr {\alpha} particles per laser shot. This constitutes presently the highest yield value normalized to the laser energy on target. The measured fusion yield per proton exceeds the classical expectation of beam-target reactions by up to four orders of magnitude under high proton intensities. This enhancement is attributed to the strong electric fields and nonequilibrium thermonuclear fusion reactions as a result of the new method. Our approach shows opportunities to pursue ignition of aneutronic fusion

A longitudinal resource for population neuroscience of school-age children and adolescents in China

During the past decade, cognitive neuroscience has been calling for population diversity to address the challenge of validity and generalizability, ushering in a new era of population neuroscience. The developing Chinese Color Nest Project (devCCNP, 2013–2022), the first ten-year stage of the lifespan CCNP (2013–2032), is a two-stages project focusing on brain-mind development. The project aims to create and share a large-scale, longitudinal and multimodal dataset of typically developing children and adolescents (ages 6.0–17.9 at enrolment) in the Chinese population. The devCCNP houses not only phenotypes measured by demographic, biophysical, psychological and behavioural, cognitive, affective, and ocular-tracking assessments but also neurotypes measured with magnetic resonance imaging (MRI) of brain morphometry, resting-state function, naturalistic viewing function and diffusion structure. This Data Descriptor introduces the first data release of devCCNP including a total of 864 visits from 479 participants. Herein, we provided details of the experimental design, sampling strategies, and technical validation of the devCCNP resource. We demonstrate and discuss the potential of a multicohort longitudinal design to depict normative brain growth curves from the perspective of developmental population neuroscience. The devCCNP resource is shared as part of the “Chinese Data-sharing Warehouse for In-vivo Imaging Brain” in the Chinese Color Nest Project (CCNP) – Lifespan Brain-Mind Development Data Community (https://ccnp.scidb.cn) at the Science Data Bank